Throughout this application, various publications are referenced and full citations for these publications may be found in the reference at the end of the specification preceding the claims. The disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to the skilled therein as of the date of the invention described and claimed herein.
It is well known that inside the gastrointestinal tract of each human being resides a microbial flora, which is a rich ecosystem of enormous complexity containing trillions of bacteria divided into more than 400 species. In addition to the normal microbial flora (resident micro flora) already established in the gastrointestinal tract, bacteria are introduced into human body everyday as a normal part of food (transient micro flora) or as contaminants (accidental micro flora).
The bacteria in human gastrointestinal tract can be divided into two groupsxe2x80x94the probiotics and the pathogens. The probiotics are beneficial since they participate in a wide variety of positive and health promoting activities in human physiology. The pathogens are harmful as they cause diseases. Normally the probiotics prevail and predominate. The microscopic interplay between probiotics and pathogens and the balance between the beneficial and the harmful microorganisms are essential for a properly functioning digestive tract, which is in turn crucial to the optimal health of a human being. The number and efficiency of probiotics decline as people age or decrease in health.
The major benefits probiotics render to human beings are as follows:
The presence and adherence of probiotics, especially many lactic acid producing bacteria to the mucous membrane of the intestine from ileo-caecal region downward constitute a formidable natural biologic barrier for many pathogenic bacteria. The probiotics compete with disease-causing bacteria for villi attachment sites and nutrients. In addition, many kinds of acid produced by probiotics, such as lactic acid, acetic acid and benzoic acid, create an acidic environment unfriendly to harmful bacteria, viruses and yeasts.(1) The metabolic by-products of many probiotics, called bacteriocins, such as hydrogen peroxide, lactocidin, acidophilin and bulgarican, also manifest antibiotic properties and inhibit the growth of a wide spectrum of pathogens and/or potential pathogens, such as Escherichia (particularly E. coli), Klebsiella, Enterobacter, Pseudomonas, Salmonella, Serratia and Bacteroides.(2)
The metabolic products of probiotics provide many useful enzymes and volatile or non-volatile fatty acids to help digestion and absorption of certain minerals, such as phosphorus, calcium and iron.(3)
Probiotics contribute to a shorter intestinal transit time as well. This effect may be useful in some cases of constipation, particularly for the elder people.
Many resident enteric probiotics also produce certain important vitamins of B complex such as niacin (B3), pyridoxine (B6), folic acid and biotin for the needs of the host human being.
The lactic acid producing bacteria confer significant protection for human beings through various immune mechanisms, that include interactions between the microbes and the host as well as interactions among microbes.(4)
Probiotics also have powerful anti-carcinogenic features, that are active against certain tumors.(5) Epidemiological reports indicate an inverse relationship between colon cancer risk and consumption of fermented milks containing certain probiotics.(6) Bifidobacterium bifidum and Lactobacillus acidophilus have been shown in human clinical studies as being able to reduce the levels of some colonic enzymes (xcex2-glucuronidase, nitroreductase, azoreductase and glycoholic acid hydrolase), that are implicated in the conversion of procarcinogens to carcinogens such as nitrosamine or secondary bile salts.(7)
Probiotics play such an integral part in the health of human being that some leading researchers in Europe claim that as much as 80% of all degenerative diseases result from an imbalance in the intestinal flora.
Probiotics, especially certain lactic acid producing bacteria have been used to ferment food for at least 4,000 years. Without understanding the scientific basis, people used these bacteria to produce cultured food with improved preservation and different flavors and textures from the original food. A wide variety of food including sausage, ham, wine, cider, beer, sauerkraut, olives and pickles all contain beneficial bacteria. Lactic acid producing bacteria are also used for many fermented milk products from all over the world, including yogurt, cheese, butter, buttermilk, kefir and koumiss.
The health benefits associated with probiotics were first observed by Nobel prize winner Elie Metchnikoff in the early 1900""s when he hypothesized that lactobacilli were important in the intestinal flora for the health and longevity of human beings.(8) At about the same time, Tissier also showed that Bifidobacteria were the predominant flora in breast-fed infants and speculated that infant diarrhea could be treated by giving large doses of Bifidobacteria orally.(9) Since then, much research has been performed in an attempt to elucidate the mechanism of action of probiotics in the intestines on good health.
Scientific interest in probiotics has increased notably in past decades as the widespread use of antibiotics such as penicillin to suppress infection also brought many side effects. Antibiotics eliminate both harmful and beneficial bacteria at the same time and disturb the balance of normal bacteria flora in human body. Patients after long period or large amount use of antibiotics are often extremely susceptible to the secondary infection. In addition, many pathogens and/or potential pathogens have acquired immunity to antibiotics over the years. Researchers all over the world are looking for a method which can suppress disease causing bacteria without disturbing the normal microbial flora in the human body. Since more and more researches have shown that probiotics have the property to inhibit the growth of pathogens and/or potential pathogens and their levels can fluctuate and be manipulated, attempts have been made to increase the number and activity of these bacteria by consuming live cultures of selected species of bacteria in milk, yogurt, or pure cultures in powder, tablet or capsule form.
In China, strain selection on certain bacteria with health benefits was undertaken in the early 1980""s when a group of scientists at the Institute of Epidemiology and Microbiology of the Chinese Preventive Medical Academy succeeded in isolating various anaerobes from the feces of 1-5 years old healthy children. They compared the number of beneficial microbes in the feces of healthy people with that of those suffering from diarrhoeal conditions, finding that Bifidobacteria together with other Gram positive non-sporing rods and residential Gram negative bacteria such as Bacteroides were greatly reduced in number in diarrhoeal patients as compared with the normal ones.(10) Realizing the significance of their findings, these scientists next performed experiments in gnotobiotic gineapigs with the Bifidobacterium bifidum and a combination of Bifidobacterium bifidum, Lactobacillus acidophilus and Sterptococcus faecalis. Challenged with a virulent strain of Shigella flexneri 2a, the researchers found that animals implanted and colonized with Bifidobacterium bifidum exhibited marked resistance to Shigella infection whereas the controls succumbed. They also discovered that Bifidobacterium bifidum, Lactobacillus acidophilus and Streptococcus faecalis, when used together, performed a synergistic action in killing Shigella sonnei.(11) Another study, using gnotocavy dysentery model test, also showed that the protection of Bifidobacterium bifidum to animal (50%) was lower than that of the three bacteria combined (75%).(12)
In the past three decades, although much progress has been made in the field of probiotic research, problems exist. First of all, although some health benefits of probiotics are now well documented, not all of the varied hypotheses have been substantiated. The problems are in part related to the fact that most health effects of probiotics are species, even strain dependent and specific, and varying results may occur when using different strains of the same species or even different preparations of the same strain. There is also a large variability in the physiology of individuals and large populations must be studied to provide valid results. In addition, many bacteria species, such as Lactobacillus acidophilus, are not viable for a great length of storage time. The 1983 University of Wyoming survey conducted by Brennan et. al. proved that the viability claims of various manufactures did not hold true with their products. Also, a majority of the probiotics get rapidly inactivated in the human upper gastrointestinal tract, where the pH is close to 2.0 to 2.9, even before they reach the lower intestines.(13) For example, China Pat. No. CN 1,103,584 describes an oral solution comprising three lactic acid producing bacteria consisting of Bifidobacterium bifidum, Lactobacillus acidophilus and Sterptococcus faecalis. This oral solution is problematic since its stability and viable ratio of microbes are low and it is difficult to store.
Notable efforts have been made since the 1980""s on selection of probiotic strains with proven therapeutic properties and survival ability. U.S. Pat. No. 5,143,845 discloses a symbiotic mixture containing three kinds of bacteria as effective ingredients that consist essentially of lactic acid producing bacteria such as Sterptococcus faecalis, saccarificating bacteria such as Bacillus mesentericus and butyric acid producing bacteria such as Clostridium butyricum. The patent also provides an unique method for culturing these three kinds of bacteria, that comprises propagating saccarificating bacteria in a culture medium containing a protein as the source of nitrogen and a carbohydrate as the source of carbon. After filtering the culture fluid to remove the saccarificating bacteria, the filtrate is introduced to a fresh culture medium, in which lactic acid producing bacteria are cultured under aerobic conditions. Then both the lactic acid producing bacteria and the butyric acid producing bacteria are propagated under anaerobic conditions in the fresh medium containing the culture fluid from the culture of the lactic acid producing bacteria. The lactic acid producing bacteria and the butyric acid producing bacteria are segmented from the culture medium after the spore formation of butyric acid producing bacteria.
U.S. Pat. No. 4,806,368 describes a nutritional supplement tablet using a lyophilized preparation of Lactobacillus acidophilus and/or Bifidobacterium bifidus, Leuconostoc citrovorum and Propionibacterium shermanii. To enhance the viability of the lyophilized bacterial preparations in the tablet, the reducing compounds such as amino acid L-cystine are included. To protect the beneficial bacterial population in the human stomach and intestinal tract, the minerals such as calcium carbonate and magnesium oxide are incorporated in the formula. The Lecithin is employed as a natural lubricant to aid the tablet preparation. The milk derived ingredients such as acid whey powder, whey protein concentrate and enzyme digested casein are used as bacterial growth enhancers. Also, the acid whey supplies bio-available minerals, simple and complex carbohydrate substrates and lactates to the bacterial growth in the intestinal tract. Further, autolyzed yeast extract is incorporated as a stimulant to the beneficial bacteria in the gastrointestinal tract, a major source of B-vitamins and a supplier of most of the major and minor trace minerals. Although vitamins A, C, D and E are included in the formula primarily as nutritional supplements, vitamins C and E also protect the bacterial cells in the tablets together with L-cystine due to their antioxidation properties. Similarly, although the diet fiber is used in the tablets, with a major proportion, mainly as a dietary supplement, the fiber, after reconstitution, also has an exceptionally protective and stimulatory effect on the growth of the probiotics in the presence of neutralizing calcium and magnesium salts.
Another composition entitled BioPRO(copyright) provided by Vitamin Research Products, Inc. located at 3579 Hwy. 50 East, Carson City, Nev. 89701 contains three species of bacteria: Lactobacillus acidophilus DDS-1 and Bifidobacterium bifidum and streptococcus faceum, all developed at the University of Nebraska. BioPRO(copyright) takes the formulation of capsule, each containing a minimum of one billion active bacteria. In addition to the bacteria, Lactobacillus Growth Factor and Fructooligosaccharides are also included in the composition to enhance the potency of BioPRO(copyright) Fructooligosaccharides are a simple class of natural carbohydrates, that exist in small quantities in people""s daily diet. Fructooligosaccharides are non-digestible, S short-chain fructose polymers that are utilized almost exclusively by the Bifidobacteria and Lactobacteria in intestinal tract of a human being. While Fructooligosaccharides can be used to enhance the growth of beneficial bacteria, harmful bacteria such as Clostridium, Salmonella and E.Coli can not get any benefits of Fructooligosaccharides. Human clinical studies have demonstrated the stimulating effect of Fructooligosaccharides on beneficial bacteria. At the clinical trials, the number of beneficial bacteria increased up to a 10-fold after Fructooligosaccharides supplementation of 1 to 4 grams.
U.S. Pat. No. 5,501,857 discloses a nutritional and dietary composition which combines certain incompatible substances such as nutrient supplements and viable direct-fed microbials in the form of a double capsule, which includes an inner capsule and an outer capsule. The outer capsule is spaced apart from and encloses the inner capsule. The inner capsule includes a dissolvable gelatin shell and a first substance. The outer capsule includes a dissolvable gelatin shell and a second substance. One of the substances is viable gastrointestinal microorganisms and the other substance is a nutritional supplement having the property of diminishing viability of the microorganisms.
The microorganisms are selected from the group consisting of gastrointestinal bacteria, live cell yeasts, fungi and a combination thereof, wherein the bacteria are one or more of the genus Lactobacillus, Streptococcus, Pediococcus, Bifidobacterium or Propionibacterium; the live cell yeast is the genus Saccharomyces; and the fungus is the genus Aspergillus. The nutritional supplement is selected from the group consisting of vitamins, minerals and a combination thereof, wherein the vitamins include one or more of vitamin A, vitamin D, vitamin E, Vitamin B12, riboflavin, niacin, pantothenic acid, thiamine, choline, folic acid, biotin, vitamin K and vitamin C, and the minerals includes one or more of cobalt, copper, iron, manganese, zinc and selenium. The composition is shelf stable and allows substantially greater viability of the microbials by using this capsule-in-a-capsule structure. U.S. Pat. No. 5,501,857 demonstrates that the bacterial survival rate with this capsule-in-a-capsule structure after six months of preparation is nearly 500 times that of an admixture of the bacteria and nutrient supplements.
It is the object of the present invention to provide a microbe composition which: (1) exerts a control mechanism for the micro ecological balance between enteric microbes and their human host; (2) is antagonistic to pathogens and/or potential pathogens such as Salmonella, Shigella, E. coli and V. cholerae, especially when the pathogens and/or potential pathogens are resistant to various antibiotics; (3) is effective in treating various kinds and degrees of diarrhea; (4) stimulates peristaltic movement of the intestinal tract which not only prevents toxic microbial colonization and eliminates noxious microbial products but also alleviates constipation; (5) enhances immunologic function of the human host; (6) is effective in decreasing the levels of endotoxin and abnormally elevated cytokine IL-6 in the blood of the human host; (7) is effective in improving and enhancing liver function; (8) is effective in treating acute and chronic hepatitis in active stage especially when it is accompanied with endotoximia; (9) is effective in alleviating liver cirrhosis; (10) is non-toxic and has no side effects and (11) is stable, convenient to store and use.
It is also an object of the present invention to protect and enhance the viability of the beneficial bacteria in the microbe composition in lyophilized form over a long period of time.
It is another object of the present invention to provide a method to prepare the microbe composition.
The present invention relates generally to a microbe composition comprising beneficial microorganisms of new strains and particularly to a symbiotic mixture of three lactic acid producing bacteria consisting of Bifidobacterium longum 6-1, Lactobacillus acidophilus YIT 2004 and Sterptococcus faecalis YIT 0027. The invention also concerns new materials to protect the viability of the bacteria in lyophilized form. In a ddition, the invention pertains to a method for culturing the microorganisms and preparing the microbe composition. The composition helps replenish the intestine with normal biological bacteria and adjust the balance of enteric bacterial species. It is also used in the treatment of acute and chronic diarrhea, abdominal distention and constipation. In addition, the composition inhibits the growth of pathogens and/or potential pathogens in the intestines, promotes the decomposition and absorption of nutritions, provides essential vitamins, enhances the body immune system and reduces the absorption of intestinal toxin to ease the burden of the liver. The composition is useful for both human beings and animals. Accordingly, the present invention will be described in detail with respect to such fields of endeavor; however, those skilled in the art will appreciate that such description of the invention is meant to be exemplary only and should not be viewed as limitative of the full scope thereof.
This invention provides a microbe composition comprising the bacteria Bifidobacterium longum 6-1.
Bifidobacterium longum 6-1 is an anaerobic bacterium which, grows under anaerobic or slight aerobic conditions. The optimal growth temperature for Bifidobacterium longum 6-1 is 37-380C and the optimal pH value is 6.5-7.4. The strain of Bifidobacterium Longum 6-1 of this invention was developed and provided by the Institute of Epidemiology and Microbiology of the Chinese Preventive Medical-Academy.
The method for culturing and identifying Bifidobacterium longum 6-1 is as follows: dissolving 0.5 g. fresh feces selected from healthy children (1-5 years old) with 4.5 ml. PBS solution; diluting the solution with PBS solution further to a suitable concentration and plating out on the BL medium; after 72 hours of culture at the temperature of 37xc2x0 C. under anaerobic conditions, Bifidobacterium longum 6-7. forms round, a bit of protrusive, milky white, smooth and adherent colonies from 0.5 to 2 mm; Gram""s stain of Bifidobacterium longum 6-1 is positive but inhomogeneous; the strain has neither spore nor capsule nor flagellum; the mycelia are straight or curved or Y-, V- and stick type; selecting typical colony for sterile culture and aerobic test; the strain of Bifidobacterium longum 6-1 is selected and tested under microscope together with test of physiological and biochemical reaction and analysis of metabolites; the strain is identified as Bifidobacterium longum 6-1, which is in agreement with some descriptions made by Buchanan B L et al. in Bergeys Manual of Determinative Bacteriology Eighth ed. (1977).
The following table compares the physicochemical characteristics of Bifidobacterium longum and Bifidobacterium longum 6-1.
Bifidobacterium(14) longum is by far the most researched species known as being able to produce natural antibiotics, including anti-virus compounds. Staying mainly inside the colon and rectum (lower intestine), Bifidobacterium longum is effective in killing and/or inhibiting bacteria, such as Escherichia coli, S. aureus and shigella, and some yeasts and viruses that may cause certain diseases. Bifidobacterium longum helps not only to detoxify bile from which they recycle oestrogen in women but also to prevent potential toxicity from nitrites coming with food. Manufacturing various IB vitamins, Bifidobacteri@m longum has also been shown in human clinical studies as being able to reduce the levels of some colonic enzymes (xcex2-glucuronidase, nitroreductase, azoreductase and glycoholic acid hydrolase), which are implicated in the conversion of procarcinogens to carcinogens such as nitrosamine or secondary bile salts.(15)
This invention provides the isolated bacteria Bifidobacterium longum 6-1. This bacteria was deposited on Feb. 23, 1998 with the international deposit agency in the People""s Republic of China, China Center for Type Culture Collection, located on the campus of Wuhan University, Wuhan 430072, P. R. China, with the telephone No. 86-027-7882712, Fax No. 86-27-7883833, under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. The viability of the culture was tested on Feb. 26, 1998. The culture was viable on that day. Bifidobacterium longum 6-1 was accorded China Center for Type Culture Colletion (CCTCC) Number M 98003.
This invention provides a microbe composition comprising the bacteria Lactobacillus acidophilus YIT 2004.
Lactobacillus acidophilus YIT 2004 is a facultative anaerobe bacterium. The optimal growth temperature for Lactobacillus acidophilus YIT 2004 is 37-400C, but the bacteria can also grow at the temperature of 45xc2x0 C. The optimal pH value is 5.5-6.5. After 48 hours of culture under aerobic condition, Lactobacillus acidophilus YIT 2004 forms round, a bit of protrusive, milky white and smooth colonies on the BL plat. The diameter of the colonies is about 1 mm. Gram""s stain of Lactobacillus acidophilus YIT 2004 is positive. The strain has neither spore, nor capsule nor flagellum. The mycelia are short stick-like or ball stick-like with blunt round at two site of stick-like mycelia and are arranged in single or twin form.
The strain of Lactobacillus acidophilus YIT 2004 of this invention was provided by Japan Gifu pharmaceutical University.
Lactobacillus acidophilus is carefully selected for use in this invention. Extensive literature backs up the health benefits of Lactobacillus acidophilus for human being. Consuming sugars and producing lactic acid, Lactobacillus acidophilus inhabits primarily in the stomach and intestinum ileum. In vitro studies have demonstrated antagonistic activity of Lactobacillus acidophilus against a variety of pathogens and/or potential pathogens, such as Helicobacter pylori,(16) Yersini pseudotuberculosis, Salmonella typhimurium(17) and Shigella sonnei.(18) Animal studies have further shown prevention of infections produced by Escherichia coli, Listeria monocytogenes(19) and Shigella sonnei.(20) These results have been attributed to certain specific compounds produced by Lactobacillus acidophilus that include certain antimicrobial compounds such as organic acids and hydrogen peroxide, antibiotic-like compounds such as lactocidin, acidophilin and bulgarican, (21) and bacteriocins (proteins or protein complexes that exert antibacterial activity against closely related species(22) such as lactacin-B and lactacin-F).(23) As early as 1950, acidophilus had been recognized by the U.S. government as a drug, when it was found equally effective as neomycin sulfate for E. coli infections.(24) Lactobacillus acidophilus also produces certain enzymes such as protease that digest proteins and lipase, that digest fats. In addition, the bacteria have the effect of shortening intestinal transit time, improving immune response and reducing serum cholesterol levels. Since the bacteria break down lactose (milk sugar) into more easily digestible simple sugars, Lactobacillus acidophilus can treat lactose intolerance. The bacteria can also help to decrease carcinogen production since they are able to reduce the level of several colonic enzymes (0-glucuronidase, nitroreductase, azoreductase and glycoholic acid hydrolase) that are implicated in the conversion of procarcinogens to carcinogens such as nitrosamine or secondary bile salts.(25) In vitro studies have as well shown decreased mutagenic activity of heterocyclic amines (procarcinogens) in the presence of Lactobacillus acidophilus. Direct binding of procarcinogens may be responsible for this effect.(26)
This invention provides the isolated bacteria Lactobacillus acidophilus YIT 2004. This bacteria was deposited on Feb. 23, 1998 with the international deposit agency in the People""s Republic of China, China Center for Type Culture Collection, located on the campus of Wuhan University, Wuhan 430072, P. R. China, with the telephone No. 86-027-7882712, Fax No. 86-27-7883833, under the provisions of the Budapest Treaty for t he International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. The viability of the culture was tested on Feb. 26, 1998. The culture was viable on that day. Lactobacillus acidophilus YIT 2004 was accorded China Center for Type Culture Colletion (CCTCC) Number M98004.
This invention provides a microbe composition comprising the bacteria Sterptococcus faecalis YIT 0027.
Sterptococcus faecalis YIT 0027 is an facultative anaerobe bacterium. The optimal growth temperature for Sterptococcus faecalis YIT 0027 is 37-40xc2x0 C., but the bacteria can also grow well at the temperature of 45xc2x0 C. The optimal pH value is 6.5-7.6. After 24 hours of culture under aerobic condition, Sterptococcus faecalis YIT 0027 forms round, a bit of protrusive, gray white and smooth colonies on the BL plat. Gram""s stain of Sterptococcus faecalis YIT 0027 is positive. The strain ha s neither spore, capsule, nor flagellum. The mycelia are egg round, arranged in twin or short chain form. The strain of Sterptococcus faecalis YIT 0027 of this invention was also provided by Japan Gifu pharmaceutical University.
This invention provides the isolated bacteria Sterptococcus faecalis YIT 0027. This bacteria was deposited on Feb. 23, 1998 with the international deposit agency in the People""s Republic of China, China Center for Type Culture Collection, located on the campus of Wuhan University, Waliaci 430072, P. R. China, with the telephone No. 86-027-7882712, Pax No. 86-27-7883833, under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. The viability of the culture was tested on Feb. 26, 1998. The culture was viable on that day. Sterptococcus faecalis YIT 0027 was accorded China Center for Type Culture Collection (CCTCC) Number M 98005.
This invention provides a microbe composition comprising the bacteria Bifidobacterium longum 6-1 (CCTCC Number M 98003), Lactobacillus acidophilus YIT 2004 (CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027 (CCTCC Number M 98005).
This invention provides a microbe composition comprising the bacteria Bifidobacterium longum 6-1 (CCTCC Number M 98003), Lactobacillus acidophilus YIT 2004 (CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027 (CCTCC Number M 98005) with a content ratio as 1-5:1-5:1-5.
The content ratios used herein are the respective numbers of colony-forming units per gram (CFU/G) for each microbe.
This invention provides a microbe composition comprising the bacteria Bifidobacterium longum 6-1 (CCTCC Number M 98003), Lactobacillus acidophilus YIT 2004 (CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027 (CCTCC Number M 98005) with a content ratio as 5:5:3.
This invention provides a microbe composition comprising the bacteria Bifidobacterium longum 6-1 (CCTCC Number M 98003), Lactobacillus acidophilus YIT 2004 (CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027 (CCTCC Number M 98005) with a content ratio as 5:3:3.
This invention provides a microbe composition comprising the bacteria Bifidobacterium longum 6-1 (CCTCC Number M 98003), Lactobacillus acidophilus YIT 2004 (CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027 (CCTCC Number M 98005) with a content ratio as 5:3:5.
This invention provides a microbe composition comprising the bacteria Bifidobacterium longum 6-1 (CCTCC Number M 98003), Lactobacillus acidophilus YIT 2004 (CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027 (CCTCC Number M 98005) with a content ratio as 5:5:1.
This invention provides a microbe composition comprising the bacteria Bifidobacterium longum 6-1 (CCTCC Number M 98003), Lactobacillus acidophilus YIT 2004 (CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027 (CCTCC Number M 98005) with a content ratio as 5:3:1.
All three bacterium strains selected for this microbe composition have been shown as being able to survive human stomach acidity as well as propagate at physiological levels of bile salts and adhere to the intestinal epithelial cells.
The combination of three bacterium strains has an advantage over single bacterium strain preparation since each of the three bacterium strains has its characteristics and specialized roles and is synergetic with each other. Each bacterium strain will differentiate itself in the intestinal tract by finding its most favorable environment to survive the intense temperature and high concentrations of bile salts in the intestinal tract. As a result, this combination is able to control pathogenic bacteria in the entire human intestine instead of only a part of it. This combination also produces an enhanced inhibitory capacity than that of a single bacterium preparation, that brings the beneficial effects of three bacterium strains into fast and steady play.
This invention provides a microbe composition comprising the bacteria Bifidobacterium longum 6-1 (CCTCC Number M 98003), Lactobacillus acidophilus YIT 2004 (CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027 (CCTCC Number M 98005) in the form of lyophilized powder. By the term of xe2x80x9clyophilized,xe2x80x9d it is intended to include substantially any techniques or variations for preserving viable bacteria in a dry or powered form, such that the bacteria can be incorporated into the formulation of a pill, capsule, tablet, granule or suspension.
This invention provides the above microbe compositions further comprising a protective medium wherein said protective medium is 10-20% dried skim milk, 10-20% lactulose, 0.01-0.2% sodium vitamin C, 0.01-2% sodium glutamate, 0.01-2% vitamin E, 6-20% of xcex2-cyclodextrin and 6-20% starch.
This invention provides the above microbe compositions wherein said protective medium is 10-20% dried skim milk, 10-20% lactulose, 0.01-0.2% sodium vitamin C, 0.01-2% sodium glutamate, 0.01-2% vitamin E and 6-20% starch.
This invention provides the above microbe compositions wherein said protective medium is 10-20% lactulose, 0.01-0.2% sodium vitamin C, 0.01-2% vitamin E and 6-20% starch.
This invention provides a microbe composition comprising the bacteria Bifidobacterium longum 6-1 (CCTCC Number M 98003), Lactobacillus acidophilus YIT 2004 (CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027 (CCTCC Number M 98005) and a protective medium.
This invention provides a microbe composition comprising the bacteria Bifidobacterium longum 6-1 (CCTCC Number M 98003), Lactobacillus acidophilus YIT 2004 (CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027 (CCTCC Number M 98005) and a protective medium with a content ratio between bacteria and protective medium as 0.01-99.99% to 99.99-0.01%.
The following tables show the stability of lyophilized powder of Bifidobacterium longum 6-1 (CCTCC Number M 98003), Lactobacillus acidophilus YIT 2004 (CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027 (CCTCC Number M 98005). All temperatures are expressed in degrees Celsius. Microbial viability is expressed in colony-forming units per gram of source material (CFU/G).
The following tables show the stability of the microbe composition comprising Bifidobacterium longum 6-1 (CCTCC Number M 98003), Lactobacillus acidophilus YIT 2004 (CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027 (CCTCC Number M 98005) in the formulation of capsule. The microbe compositions in table 5 and 6 were prepared with zymotic fluid comprising skim milk, glucose, ammonia sulfate, monopotassium phosphate, dipotassium hydrogen phosphate and calcium carbonate and with protective materials comprising skim milk, lactulose, sodium vitamin C and sodium glutamate. The microbe composition in table 7 was prepared with zymotic fluid comprising skim milk, glucose, yeast powder, ammonia sulfate, monopotassium phosphate, dipotassium hydrogen phosphate and calcium carbonate and with protective materials comprising skim milk, lactulose, sodium vitamin C, sodium glutamate, vitamin E, xcex2-cyclodextrin and compressible starch. All temperatures are expressed in degrees Celsius. Microbial viability is expressed in colony-forming units per gram of source material (CFU/G).
The total viable bacteria is 1.9-6.5 hundred million CFU/g. when produced and 6.7-30 million CFU/g. after storage in refrigerator for one year.
This invention provides a method for preparing a microbe composition comprising steps of: (a) mixing solutions of 3.5-4% dried skim milk, 1% glucose and 3% yeast extract; (b) sterilizing the mixed solution in a autoclave at a temperature of 1210C for 10 minutes, then allowing sterilized medium to cool to 37-380C and transferring the medium to three fermentation tanks; (c) inoculating Bifidobacterium longum 6-1 (CCTCC Number M 98003), Lactobacillus acidophilus YIT 2004 (CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027 (CCTCC Number M 98005) with 10% seed respectively into different fermentation tank; (d) culturing Bifidobacterium longum 6-1 (CCTCC Number M 98003) at a temperature of 37-38xc2x0 C., a pressure of 0.3-0.6 kg/cm (inner) under anaerobic and nitrogenous conditions; culturing Lactobacillus acidophilus YIT 2004(CCTCC Number M 98004) and Sterptococcus faecalis YIT 0027(CCTCC Number M 98005) at the same temperature and pressure but with sterile gas; (e) separating the bacteria from the medium by centrifugation at 20000 rpm; (f) adding same amount of protective medium to the bacteria mud; (g) lyophilizing the composition; (h) mixing the lyophilized power with starch and pre-glued starch; and (I) preparing the lactic acid producing bacteria composition in forms of capsule, tablet or granule.
This invention provides the above methods for preparing a microbe composition wherein said protective medium comprises 10-20% dried skim milk, 10-20% lactulose, 0.01-0.2% sodium vitamin C, 0.01-2% sodium glutamate, 0.01-2% vitamin E, 6-20% xcex2-cyclodextrin and 6-20% starch.
This invention provides the above methods for preparing a microbe composition wherein said protective medium comprises 10-20% dried skim milk, 10-20% lactulose, 0.01-0.2% sodium vitamin C, 0.01-2% sodium glutamate, 0.01-2% vitamin E and 6-20% starch.
This invention provides the above methods for preparing a microbe composition wherein said protective medium comprises 10-20% lactulose, 0.01-0.2% sodium vitamin C, 0.01-2% vitamin E and 6-20% starch.
This invention provides an zymotic fluid comprising skim milk, glucose, ammonia sulfate, monopotassium phosphate, dipotassium hydrogen phosphate and calcium carbonate.
This invention provides an zymotic fluid comprising skim milk, glucose, yeast powder, ammonia sulfate, monopotassium phosphate, dipotassium hydrogen phosphate and calcium carbonate.
This invention provides an oral formulation containing the above microbe compositions.
This invention provides the above oral formulation that can take the form of pill, capsule, tablet, granule or a suspension.
This invention provides a method for balancing intestinal flora in a subject comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for inhibiting the growth of Shigella sonnei and Shigella flexneri comprising contacting an effective amount of the above microbe compositions with Shigella sonnei and Shigella flexneri. 
This invention provides a method for treating various kinds and degrees of diarrhea in a subject comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for treating constipation in a subject comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for regulating and enhancing immunologic function in a subject by increasing the serum complement 3 (C3) level comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for regulating and enhancing immunologic function in a subject by promoting specific antibody formation comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for regulating and enhancing immunologic function in a subject by activating proliferation of B and T lymphocytes comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for decreasing the level of plasma endotoxin in a subject comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for decreasing the level of abnormally elevated cytokine IL-6 in a subject comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for improving and enhancing liver function by decreasing serum alanine transaminase (ALT) level in a subject comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for improving and enhancing liver function by decreasing serum AUT level in a subject comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for improving and enhancing liver function by decreasing serum AST level in a subject comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for improving and enhancing liver function by decreasing serum bilirubin (SB) level in a subject comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for improving and enhancing liver function by decreasing serum globulin level in a subject comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for improving and enhancing liver function by increasing serum albumin level in a subject comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for treating acute hepatitis in active stage in a subject, especially when it is accompanied with endotoximia comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for treating chronic hepatitis in active stage in a subject, especially when it is accompanied with endotoximia comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for alleviating liver injuries including swelling, degeneration and necrosis in a subject comprising administering to the subject an effective amount of the above microbe compositions.
This invention provides a method for alleviating liver cirrhosis in a subject comprising administering to the subject an effective amount of the above microbe compositions.